The present invention relates to the separation and detection of common anionic species using capillary electrophoresis, and more particularly to the use of temperature control in a capillary electrophoresis system to improve separation and reproducibility for the detection of such anionic species.
The separation and/or detection of ionic species in chemical analysis is generally carried out using the electrochemical properties of the analytes. These properties may include ionic interactions and conductivity in ion chromatography and ionic mobility in capillary electrophoresis. Capillary zone electrophoresis (CZE) is a powerful and efficient method to separate small analytes at very low concentration levels by exploiting the different mobilities of sample components in an electric field.
A conventional CZE system typically includes a buffer-filled capillary column with inlet and outlet ends disposed into two reservoirs. The buffer is generally an electrically conductive medium, sometimes termed the "carrier electrolyte". The system also includes means for introducing the sample into the capillary column, an on-column detector for sensing the sample zones as they pass the detector, and a high voltage source to apply a voltage to the capillary column to cause migration and separation of the sample into identifiable components. The ionic species in the sample move from one electrode toward the other in the capillary column at a rate which is dependent, inter alia, upon the electrical charge, molecular size, mobility of those ions, and/or field strength.
However, many analytes, including most inorganic ions, do not absorb ultraviolet or visible light. As capillary electrophoresis systems generally use direct photometric techniques such as ultraviolet/visible light (UV-VIS) detectors, the ions pass by the detector without being observed. These ions can be detected, however, using the technique of indirect photometric detection. Indirect photometric detection relies on the presence of light-absorbing buffer electrolyte ions in the sample. It is the absorbance of these buffer electrolytes which is monitored by the detector, not the absorptivity that the sample components may display.
Because the solution in the capillary is constrained to remain electrically neutral, sample ions displace the light-absorbing buffer electrolyte ions on a charge-for-charge basis as the sample migrates through the capillary. As the buffer electrolyte ions are displaced by the sample ions, more photons pass through to the detector. This increase in light throughput is recorded by the detector as a decrease in absorbance and is characterized by a negative peak. The magnitude of the negative peak is dependent upon the concentration of the displacing ion, the ratio of the negative charges on the buffer electrolyte ion to the sample ion, and finally, the concentration and extinction coefficient of the buffer electrolyte. Thus, non-absorbing ion species in the sample can be detected, and their concentrations determined using this technique.
Methods using indirect photometric detection in capillary electrophoresis have been described in the published literature, for example, by Foret et al., J. Chromatography, 470:299-308 (1989), and Kuhr et al., Anal. Chem., 60:2642-2646 (1988) and 60:1832-1844 (1988). Jones et al., U.S. Pat. Nos. 5,104,506, 5,128,005, and 5,156,724, also describe the use of indirect photometric techniques for the separation and detection of samples containing mixtures of common ionic species. However, problems remain in attempting to resolve and identify certain ion mixtures because some ions have similar migration rates and transparency to or absorbance of ultraviolet light.
To improve sensitivities for detection of certain ions below concentrations of about 1 ppm, other techniques such as electrokinetic injection, the use of electroosmotic flow modifiers, and changes in pH have been used. However, the need still exists in the art for improved methods in detection techniques in capillary electrophoresis, both to improve the separation and detection of very small concentrations of ion species, especially those which are nonabsorbers of light.